The major goals of this proposal are to better understand early hematopoietic development and to develop the potential of murine embryonic stem (ES) cells as an in vitro source of hematopoietic stem cells capable of reconstituting lethally irradiated animals. Embryonic stem cells are pluripotent tissue culture cell lines derived from early blastocysts. These cells are truly pluripotent and have the capacity to differentiate, in vitro, into many different cell types including hematopoietic cells. If implanted back into a developing mouse embryo, ES cells will participate in the normal development of all tissues. This capacity allows powerful new approaches to studying hematopoietic development and bone marrow transplantation which are not currently feasible. We have strong evidence that ES cells are capable of differentiating, in vitro, into all of the colony forming cell types found in normal bone marrow. In addition we have data on the expression of several interleukins and their receptors during the differentiation of ES cells into hematopoietic cells in vitro. The immediate goals of this grant are to characterize the in vitro hematopoietic development of ES cells and determine what growth factors or cytokines are critical for this differentiation. We will also work toward developing ES cells as an in vitro source of stem cells capable of reconstituting lethally irradiated animals. The following four aims are proposed: 1) to characterize the hematopoietic cells which develop in vitro during ES cell differentiation; 2) to determine if ES cells or their progeny are able to reconstitute lethally irradiated mice; 3) to determine the growth factors and/or conditions which are required and/or favor hematopoietic cell development from ES cells; 4) to evaluate current established culture conditions for their ability to maintain and possibly expand hematopoietic stem cells from ES cells as well as from normal bone marrow.